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Background Bone-modifying agent (BMA) therapy is recommended for metastatic castration-resistant prostate cancer but not metastatic castration-sensitive prostate cancer (mCSPC). BMA treatment in mCSPC may therefore constitute overuse. Methods In this retrospective cohort study using linked Surveillance, Epidemiology, and End Results–Medicare data, we included patients diagnosed with stage IV prostate adenocarcinoma from 2007 to 2015 who were 66 years of age or older at diagnosis and had received androgen-deprivation or antiandrogen therapy. We excluded patients who had previously received BMAs or had existing osteoporosis, osteopenia, hypercalcemia, or prior bone fracture. The primary outcome was receipt of BMA (zoledronic acid or denosumab) within 180 days of diagnosis (emergence of CRPC within this time frame is unlikely). The secondary outcome was receipt of a BMA within 90 days. Exposures of interest included practice location (physician office vs hospital outpatient) and the specialty (medical oncologist vs urologist) of the treating physician. Results Our sample included 2627 patients, of whom 52.9% were treated by medical oncologists and 47.1% by urologists; 77.7% and 22.3% received care in physician office and hospital outpatient locations, respectively. Overall, 23.6% received a BMA within 180 days; 18.4% did within 90 days. BMA therapy was more common among patients treated by oncologists (odds ratio = 8.23, 95% confidence interval = 6.41 to 10.57) and in physician office locations (odds ratio = 1.33, 95% confidence interval = 1.06 to 1.69). Utilization has increased: 17.3% of patients received BMAs from 2007 to 2009 (17.3% zoledronic acid, 0% denosumab) and 28.1% from 2012 to 2015 (8.4% zoledronic acid, 20.3% denosumab). Conclusions Among patients with mCSPC who had no evidence of high osteoporotic fracture risk, more than one-quarter have received BMAs in recent years. This overuse may lead to excess costs and toxicity.

BACKGROUND: Financial relationships between physicians and the pharmaceutical and medical device industries are common, but the factors associated with physicians receiving payments are unknown. OBJECTIVE: The objective of this study is to evaluate the influence of physicians’ professional networks’ characteristics on the receipt of payments among physicians. DESIGN: Network analysis of cross-sectional data PARTICIPANTS: US physicians who shared Medicare patients with other physicians in 2015 (N=357,813). EXPOSURE (INTERVENTION): Proportion of a physician’s professional network that received industry payments and other network characteristics including number of physician connections, how central the physician is within the network, and the tightness of the referral network in which a physician is located. MAIN OUTCOME MEASURES: Relative risk of receiving industry payments. We used modified Poisson regression to control for confounding by gender, time since graduation, practice size, and practice setting (teaching hospital vs. not). We included dummy variables for specialty and hospital referral region level. KEY RESULTS: The proportion of a physician’s peers in their professional network that received payments was strongly associated with receipt of pharmaceutical or device industry payments by the physician (top vs bottom quartile aRR=1.28, 95%CI=1.25–1.31). Physician’s centrality within a network had a small positive effect on receiving payment (top vs bottom quartile aRR=1.02, 95%CI=1.01–1.04). Network density also had a small negative association with receipt of payment (top vs bottom quartile aRR=0.97, 95%CI=0.96–0.98). CONCLUSIONS: Network characteristics, particularly the receipt of payments among physicians one shares patients with, are associated with whether a physician receives payments. This finding has implications for institutional regulation of industry payments to physicians and demonstrates how institutional policy may impact not only the physicians within the institution but also physicians outside of the institution. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11606-021-06802-9.

Simple Summary Enfortumab Vedotin (EV) is FDA-approved for advanced urothelial cancer in patients previously treated with chemotherapy and immunotherapy. In this report, we looked at the extent of EV wastage (i.e., discarding of leftover drug not administered to the patient) in a single institute and estimated the financial impact of EV wastage annually in the United States. We found that wastage occurred in 46% of administered doses, with an average waste per dose of 2.9% (range 0–18%). The average drug wastage cost per patient was $3127 ($252 per dose). The annual cost of EV wastage in the US is estimated to be $15 million. Abstract Enfortumab Vedotin (EV) is FDA-approved for advanced urothelial cancer in patients previously treated with platinum-based chemotherapy and a checkpoint inhibitor. We conducted a real-world study to determine the extent of EV wastage in a single institution and assessed the financial impact of EV wastage annually in the United States. Systematic examination of the usage and wastage of all standard-of-care EV treatments administered to urothelial cancer patients at Memorial Sloan Kettering Cancer Center (MSKCC) between 1 January 2020 and 31 December 2020 was performed. Drug wastage was calculated by subtracting the actual administered dose from the total dose in an optimal set of vials. We built a pharmacoeconomic model to assess the financial impact of EV wastage annually in the US using the January 2021 Average Sales Prices from the Centers for Medicare and Medicaid Services. Sixty-four patients were treated with standard-of-care EV, with a median of 11 doses per patient (range 1–28). Wastage occurred in 46% of administered doses (367/793), with a mean waste per dose of 2.9% (0–18%). The average drug wastage cost per patient was $3127 ($252/dose). The annual cost of EV wastage in the US is estimated to be $15 million based on wastage data from a single center in the US. In summary, EV wastage due to available vial sizes was 2.9%, which falls under acceptable thresholds. While the percentage of EV wastage is relatively low, waste-minimizing practices may reduce the financial toxicity for the individual patient and for society.

Background: NCCN produces highly influential disease-specific oncology clinical practice guidelines. Because the number of women in academic oncology has increased, we assessed whether the composition of NCCN Guidelines Panels reflected this trend. Methods: Using historical guidelines requested from NCCN, we investigated time trends for female representation on 21 NCCN Guidelines Panels and analyzed the trends for female-predominant cancers (breast, ovarian, uterine, and cervical) compared with all cancers. Results: From 2013 to 2019, there was an increase from 123 women of 541 total panelists (22.7%) to 175 women of 542 panelists (32.3%). Within the 4 female-predominant cancers, the increase was more rapid: from 30 of 101 total panelists (29.7%) to 66 of 118 panelists (56.4%). Excluding female-predominant cancers, increases were minimal. Conclusions: There could be multiple explanations for these differing trends, including the possibility of more rapid increases in the underlying pool of female physician-scientists in female-predominant specialties or more efforts to increase the representation of women in decisions about the standard of care in cancers predominantly affecting women.

Background Oncologists who author clinical practice guidelines frequently have financial relationships with the pharmaceutical industry. It is unknown whether participation on clinical practice guideline committees is associated with differences in the amounts of industry money received. Materials and Methods We conducted a nested case-control study from August 2013 to December 2018. We manually abstracted membership records of National Comprehensive Cancer Network (NCCN) Guidelines committees for the 20 most common cancers and linked to Open Payments. The study sample included medical oncologists selected to join an NCCN Guidelines committee (“joiners”) during the study period. Joiners were matched 1:2 to medical oncologists who had no participation on NCCN committees (controls) by gender, NCCN institution, and medical school graduation year. We performed difference-in-differences (DiD) estimation to assess whether selection to an NCCN committee was associated with the dollar value of payments received from industry, using generalized estimating equations to address correlation between matched pairs and between repeated observations of the same pair. Results During the study period, 54 physicians joined an NCCN Guidelines committee. These physicians received more payments than matched controls in the year prior to joining ($11,259 vs. $3,427; p = .02); this difference did not increase in the year after joining (DiD = $731; p = .45). Conclusion Medical oncologists selected to NCCN Guidelines committees had greater financial ties to industry than their peers. The potential influence of industry in oncology clinical practice guidelines may be reduced through the selection of committee members with fewer ties to industry. Implications for Practice Oncologists who author clinical practice guidelines frequently have financial conflicts of interest with the pharmaceutical industry. This creates concern about the potential for industry influence on guidelines. However, it is unknown whether oncologists who author guidelines have greater industry relationships than their peers. This study compared medical oncologists who were newly selected to join a National Comprehensive Cancer Network (NCCN) Guidelines panel with medical oncologists at the same institutions and at similar career stages. At the time they joined, oncologists joining NCCN Guidelines panels had received more than three times the dollar value of industry payments than their peers. The potential for industry influence may be reduced by the selection of less-conflicted panel members.

The fungal pathogen Candida albicans is surrounded by a cell wall that is the target of caspofungin and other echinocandin antifungals. Candida albicans can grow in several morphological forms, notably budding yeast and hyphae. Yeast and hyphal forms differ in cell wall composition, leading us to hypothesize that there may be distinct genes required for yeast and hyphal responses to caspofungin. Mutants in 27 genes reported previously to be caspofungin hypersensitive under yeast growth conditions were all caspofungin hypersensitive under hyphal growth conditions as well. However, a screen of mutants defective in transcription factor genes revealed that Cup9 is required for normal caspofungin tolerance under hyphal and not yeast growth conditions. In a hyphal-defective efg1Δ/Δ background, Cup9 is still required for normal caspofungin tolerance. This result argues that Cup9 function is related to growth conditions rather than cell morphology. RNA-seq conducted under hyphal growth conditions indicated that 361 genes were up-regulated and 145 genes were down-regulated in response to caspofungin treatment. Both classes of caspofungin-responsive genes were enriched for cell wall-related proteins, as expected for a response to disruption of cell wall integrity and biosynthesis. The cup9Δ/Δ mutant, treated with caspofungin, had reduced RNA levels of 40 caspofungin up-regulated genes, and had increased RNA levels of 8 caspofungin down-regulated genes, an indication that Cup9 has a narrow rather than global role in the cell wall integrity response. Five Cup9-activated surface-protein genes have roles in cell wall integrity, based on mutant analysis published previously (PGA31 and IFF11) or shown here (ORF19.3499, ORF19.851, or PGA28), and therefore may explain the hypersensitivity of the cup9Δ/Δmutant to caspofungin. Our findings define Cup9 as a new determinant of caspofungin susceptibility.

In the current issue of Arthritis & Rheumatology, Putman et al report findings regarding the distribution and temporal trends of industry payments to US rheumatologists [cite ar-21-0182]. Their findings are in line with similar reports from other medical and surgical specialties, and highlight the overarching concern regarding the ability of industry payments to adversely affect care quality within the specific context of rheumatology practice.

During oropharyngeal candidiasis (OPC), Candida albicans invades and damages oral epithelial cells, which respond by producing proinflammatory mediators that recruit phagocytes to foci of infection. The ephrin type-A receptor 2 (EphA2) detects β-glucan and plays a central role in stimulating epithelial cells to release proinflammatory mediators during OPC. The epidermal growth factor receptor (EGFR) also interacts with C. albicans and is known to be activated by the Als3 adhesin/invasin and the candidalysin pore-forming toxin. Here, we investigated the interactions among EphA2, EGFR, Als3 and candidalysin during OPC. We found that EGFR and EphA2 constitutively associate with each other as part of a heteromeric physical complex and are mutually dependent for C. albicans-induced activation. Als3-mediated endocytosis of a C. albicans hypha leads to the formation of an endocytic vacuole where candidalysin accumulates at high concentration. Thus, Als3 potentiates targeting of candidalysin, and both Als3 and candidalysin are required for C. albicans to cause maximal damage to oral epithelial cells, sustain activation of EphA2 and EGFR, and stimulate pro-inflammatory cytokine and chemokine secretion. In the mouse model of OPC, C. albicans-induced production of CXCL1/KC and CCL20 is dependent on the presence of candidalysin and EGFR, but independent of Als3. The production of IL-1α and IL-17A also requires candidalysin but is independent of Als3 and EGFR. The production of TNFα requires Als1, Als3, and candidalysin. Collectively, these results delineate the complex interplay among host cell receptors EphA2 and EGFR and C. albicans virulence factors Als1, Als3 and candidalysin during the induction of OPC and the resulting oral inflammatory response., Author summary Oropharyngeal candidiasis occurs when the fungus Candida albicans proliferates in the mouth to a point at which tissue damage occurs. The disease is characterized by fungal invasion of the superficial epithelium and a localized inflammatory response. Two C. albicans virulence factors contribute to the pathogenesis of OPC, Als3 which enables the organism to adhere to and invade host cells, and candidalysin which is a pore-forming toxin that damages host cells. Two epithelial cell receptors, ephrin type-A receptor 2 (EphA2) and the epidermal growth factor receptor (EGFR) are activated by C. albicans. Here, we show that EphA2 and EGFR form part of complex wherein these co-receptors are required to activate each other. Als3 enhances the host cell targeting of candidalysin by stimulating epithelial cell endocytosis of C. albicans, leading to the formation of an endocytic vacuole in which candidalysin accumulates. Thus, Als3 and candidalysin synergize to damage epithelial cells, activate EphA2 and EGFR, and stimulate the production of inflammatory mediators. In the mouse model of OPC, candidalysin elicits of a subset of the oral inflammatory response molecular repertoire. Of the cytokines and chemokines induced by this toxin, some require the activation of EGFR while others are induced independently of EGFR. Collectively, this work provides a deeper understanding of the interactions among C. albicans virulence factors, host cell receptors and immune responses during OPC.

Among 84,447 radiotherapy (RT) courses for Medicare beneficiaries age≥65 with prostate cancer treated with external beam RT (EBRT), brachytherapy, or both, 42,608 (51%) were delivered in hospital-affiliated and 41,695 (49%) in freestanding facilities. Freestanding centers were less likely to use EBRT+brachytherapy than EBRT (OR 0.84 [95%CI 0.84–0.84];p

Background Financial payments from the drug industry to U.S. physicians are common. Payments may influence physicians' clinical decision making and drug prescribing. Purpose To evaluate whether receipt of payments from the drug industry is associated with physician prescribing practices. Data sources MEDLINE (Ovid), Embase, the Cochrane Library, Web of Science, and EconLit were searched without language restrictions. The search had no limiting start date and concluded on 16 September 2020. Study selection Studies that estimated the association between receipt of industry payments (exposure) and prescribing (outcome). Data extraction Pairs of reviewers extracted the primary analysis or analyses from each study and evaluated risk of bias (ROB). Data synthesis Thirty-six studies comprising 101 analyses were included. Most studies (n = 30) identified a positive association between payments and prescribing in all analyses; the remainder (n = 6) had a mix of positive and null findings. No study had only null findings. Of 101 individual analyses, 89 identified a positive association. Payments were associated with increased prescribing of the paying company's drug, increased prescribing costs, and increased prescribing of branded drugs. Nine studies assessed and found evidence of a temporal association; 25 assessed and found evidence of a dose-response relationship. Limitation The design was observational, 21 of 36 studies had serious ROB, and publication bias was possible. Conclusion The association between industry payments and physician prescribing was consistent across all studies that have evaluated this association. Findings regarding a temporal association and dose-response suggest a causal relationship. Primary funding source National Cancer Institute.

Transcriptional analysis has become a cornerstone of biological research, and with the advent of cheaper and more efficient sequencing technology over the last decade, there exists a need for high-yield and efficient RNA extraction techniques. Fungi such as the human pathogen Candida albicans present a unique obstacle to RNA purification in the form of the tough cell wall made up of many different components such as chitin that are resistant to many common mammalian or bacterial cell lysis methods. Typical in vitro C. albicans cell harvesting methods can be time consuming and expensive if many samples are being processed with multiple opportunities for product loss or sample variation. Harvesting cells via vacuum filtration rather than centrifugation cuts down on time before the cells are frozen and therefore the available time for the RNA expression profile to change. Vacuum filtration is preferred for C. albicans for two main reasons: cell lysis is faster on non-pelleted cells due to increased exposed surface area, and filamentous cells are difficult to pellet in the first place unlike yeast or bacterial cells. Using mechanical cell lysis, by way of zirconia/silica beads, cuts down on time for processing as well as overall cost compared to enzymatic treatments. Overall, this method is a fast, efficient, and high-yield way to extract total RNA from in vitro cultures of C. albicans.

Background: The cost of cancer treatment has increased significantly in recent decades, but it is unclear whether these costs have been associated with commensurate improvement in clinical value. This study aimed to assess the association between the cost of cancer treatment and 4 of the 5 NCCN Evidence Blocks (EB) measures of clinical value: efficacy of regimen/agent, safety of regimen/agent, quality of evidence, and consistency of evidence. Methods: This is a cross-sectional, observational study. We obtained NCCN EB ratings for all recommended, first-line, and/or maintenance treatments for the 30 most prevalent cancers in the United States and calculated direct pharmacologic treatment costs (drug acquisition, administration fees, guideline-concordant supportive care medications) using Medicare reimbursement rates in January 2019. We used generalized estimating equations to estimate the association between NCCN EB measures and treatment cost with clustering at the level of the treatment indication. Results: A total of 1,386 treatments were included. Among time-unlimited treatments (those administered on an ongoing basis without a predetermined stopping point), monthly cost was positively associated with efficacy ($3,036; 95% CI, $1,782 to $4,289) and quality of evidence ($1,509; 95% CI, $171 to $2,847) but negatively associated with safety (–$1,470; 95% CI, –$2,790 to –$151) and consistency of evidence (–$2,003; 95% CI, –$3,420 to –$586). Among time-limited treatments (those administered for a predetermined interval or number of cycles), no NCCN EB measure was significantly associated with treatment cost. Conclusions: An association between NCCN EB measures and treatment cost was inconsistent, and the magnitude of the association was small compared with the degree of cost variation among treatments with the same EB scores. The clinical value of cancer treatments does not seem to be a primary determinant of treatment cost.

The National Comprehensive Cancer Network (NCCN) Guidelines’ Evidence Blocks has the broadest scope of the several oncology value assessment frameworks. The Evidence Blocks includes the Affordability criterion, which reflects the financial cost of each treatment on a 1–5 scale. The accuracy of Affordability is unknown. We calculated Medicare costs for all first-line and maintenance treatments for the 30 cancers with the highest incidence in the USA that had published NCCN Evidence Blocks as of 31 December 2018. We assessed the accuracy and consistency of Affordability across different treatments and cancer types. Among different treatments for the same indication, we determined the frequency with which the Affordability assessment was consistent with calculated treatment costs. There were a total of 1386 treatments in our sample. Lower Affordability scores were associated with higher costs. There was significant variation in cost at each level of Affordability; for treatments with Affordability = 1 (very expensive), costs ranged from $US4551 to $US43,794 per month for treatments administered over an undefined time period and from $US2865 to $US500,982 per course of therapy for treatments administered over a defined time period. Among treatments for the same indication, Affordability was discrepant with calculated treatment costs in 7.9% of pairwise comparisons, identifying the higher-cost treatment as being more affordable. Discrepancies were reduced when we reassigned Affordability scores based on calculated treatment costs. Evidence Blocks Affordability generally correlated with treatment costs but contained discrepancies, which may limit its usefulness to clinicians in comparing costs. This study suggests that the Affordability score may be improved by indexing more directly to specified dollar value thresholds.

Prior research demonstrated statistically significant racial disparities related to lung cancer treatment and outcomes. We examined differences in initial imaging and survival between blacks, Hispanics, and non-Hispanic whites.The linked Surveillance, Epidemiology, and End Results-Medicare database between 2007 and 2015 was used to compare initial imaging modality for patients with lung cancer. Participants included 28 881 non-Hispanic whites, 3123 black, and 1907 Hispanics, patients age 66 years and older who were enrolled in Medicare fee-for-service and diagnosed with lung cancer. The primary outcome was comparison of positron emission tomography (PET) imaging with computerized tomography (CT) imaging use between groups. A secondary outcome was 12-month cancer-specific survival. Information on stage, treatment, and treatment facility was included in the analysis. Chi-square test and logistic regression were used to evaluate factors associated with imaging use. Kaplan-Meier method and Cox proportional hazards regression were used to calculate adjusted hazard ratios and survival. All statistical tests were two-sided.After adjusting for demographic, community, and facility characteristics, blacks were less likely to undergo PET or CT imaging at diagnosis compared with non-Hispanic whites odds ratio (OR) = 0.54 (95% confidence interval [CI] = 0.50 to 0.59; P .001). Hispanics were also less likely to receive PET with CT imaging (OR = 0.72, 95% CI = 0.65 to 0.81; P .001). PET with CT was associated with improved survival (HR = 0.61, 95% CI = 0.57 to 0.65; P .001).Blacks and Hispanics are less likely to undergo guideline-recommended PET with CT imaging at diagnosis of lung cancer, which may partially explain differences in survival. Awareness of this issue will allow for future interventions aimed at reducing this disparity.

Background Asplenic patients are at risk for severe infections, but adherence to recommended preventive education and vaccination is poor. The goal of this study was to demonstrate that a targeted intervention can improve vaccination rates in a population of asplenic veterans. Methods Surgically asplenic patients actively receiving care in our health care system were identified via a database search. Patients were contacted via mailed letters and encouraged to attend an existing travel clinic with a new process designed for asplenic patients. In the clinic, patients were educated on the risks of asplenia and proper preventive precautions, a vaccination history was taken, and patients were administered any additional indicated vaccines. Results The database search yielded 113 patients; an additional 14 asplenic patients were identified and referred to the clinic by providers, and 2 were referred prior to planned splenectomy. Among all asplenic patients, the first-year referral rate to clinic was 38/129 (29%). During the first year of the intervention, there were increases in the rates of 3 of 4 recommended vaccinations: pneumococcal conjugate, 19% to 55% ( P Haemophilus influenzae type B, 19% to 35% ( P = .007); and meningococcal vaccine, 24% to 43% ( P = .002). The pneumococcal polysaccharide vaccination rate increased from 91% to 93% ( P = .62). Conclusions Targeted interventions can improve guideline-based care for asplenic patients. The creation of a clinic designed for asplenic patients led to increases in 3 of 4 recommended vaccinations. This strategy may be applicable to other health care systems with similar numbers of asplenic patients.

Outcomes for metastatic colorectal cancer have improved progressively with the incorporation of new drugs into standard treatment regimens. Most recently, targeted therapies against VEGF and EGFR have improved upon the prior standard for first-line therapy with FOLFOX or FOLFIRI. As attempts to combine anti-VEGF and anti-EGFR drugs have been unsuccessful, it is necessary to choose between them when beginning first-line therapy. This review summarizes the existing literature to best inform this decision. To date, three head-to-head trials have compared anti-EGFR and anti-VEGF therapy in RAS wild-type patients: PEAK, FIRE-3, and CALGB/SWOG 80405. PEAK and FIRE-3 suggested a survival advantage for anti-EGFR therapy over anti-VEGF therapy, though CALGB/SWOG 80405 did not. Results have emerged recently to suggest that tumors arising from the right colon are resistant to anti-EGFR therapy, and that any advantage of anti-EGFR therapy over anti-VEGF therapy may be limited to left-sided tumors.

Background Academic physicians, such as those affiliated with National Cancer Institute (NCI)–designated Comprehensive Cancer Centers, may have different practice patterns regarding the use of high-cost cancer drugs than nonacademic physicians. Materials and Methods For this cohort study, we linked cancer registry, administrative, and demographic data for patients with newly diagnosed cancer in North Carolina from 2004 to 2011. We selected cancer types with multiple U.S. Food and Drug Administration–approved, National Comprehensive Cancer Network–recommended treatment options and large differences in reimbursement between higher-priced and lower-priced options (stage IV colorectal, stage IV lung, and stage II–IV head-and-neck cancers). We assessed whether provider's practice setting—NCI-designated Comprehensive Cancer Center (“NCI”) versus other location (“non-NCI”)—was associated with use of higher-cost treatment options. We used inverse probability of exposure weighting to control for patient characteristics. Results Of 800 eligible patients, 79.6% were treated in non-NCI settings. Patients treated in non-NCI settings were more likely to receive high-cost treatment than patients treated in NCI settings (36.0% vs. 23.2%), with an unadjusted prevalence difference of 12.7% (95% confidence interval [CI], 5.1%–20.0%). After controlling for potential confounding factors, non-NCI patients remained more likely to receive high-cost treatment, although the strength of association was attenuated (adjusted prevalence difference, 9.6%; 95% CI −0.1%–18.7%). Exploratory analyses suggested potential heterogeneity across cancer type and insurance status. Conclusion Use of higher-cost cancer treatments may be more common in non-NCI than NCI settings. This may reflect differential implementation of clinical evidence, local practice variation, or possibly a response to the reimbursement incentives presented by chemotherapy billing.

68 Background: Bone modifying agents (BMAs) do not prevent skeletal related events (SREs) among patients with metastatic, castration-sensitive prostate cancer (mCSPC). Within this population, BMAs are recommended for prevention of osteoporotic fractures but not for SRE prevention. Utilization patterns of BMAs among patients with mCSPC have not been described, and it is unknown whether these patients may receive BMAs intended for SRE prevention unnecessarily. We conducted this study to measure BMA use among mCSPC patients who are unlikely to have an indication for osteoporotic fracture prevention. Methods: We used linked SEER-Medicare data. Our cohort included men newly diagnosed with de-novo stage IV prostate adenocarcinoma during 2007-2015. We included those age > = 66 at diagnosis, who lived at least 60 days after diagnosis, had continuous enrollment in Medicare Parts A and B from 180 days prior to diagnosis through the outcome period and Part D from diagnosis through outcome period, and who received androgen deprivation or antiandrogen therapy after diagnosis. We excluded patients who had previously received BMAs, or who had existing osteoporosis, osteopenia, hypercalcemia, or prior bone fracture. Our primary outcome was receipt of a BMA (zoledronic acid or denosumab) within 180 days of prostate cancer diagnosis, during which time the development of castrate-resistant disease is unlikely to arise. We also assessed receipt of BMAs within 90 days of diagnosis as a secondary outcome. We used multivariable logistic regression to assess patient factors associated with BMA use. Results: Our sample included 2,998 patients. Median age at diagnosis was 77. 77% of patients were white, 12% were Black, and 11% were of other race/ethnicity. 77% of patients were treated in the physician office setting and 23% the hospital outpatient setting. Overall, 24% received a BMA within 180 days of diagnosis; 18% did within 90 days. BMA therapy was more common among patients treated in the hospital outpatient vs. physician office setting (OR 1.25, 95%CI 1.03-1.52). Age, race/ethnicity, comorbidity, and renal disease were not associated with likelihood of BMA therapy. There was substantial variation across the study period: 19% of patients received BMAs from 2007-2009 (19% zoledronic acid, 0% denosumab), and 29% from 2012-2015 (9% zoledronic acid, 21% denosumab). Conclusions: Use of BMAs among patients with mCSPC is common. In this cohort of mCSPC patients, who were unlikely to have a BMA indication for osteoporotic fracture prevention based on the absence of history of osteoporosis, osteopenia, or fracture, nearly one-quarter received BMAs. The increase in BMA use which occurred during the 2007-2015 study period appears to have been driven by increased utilization of denosumab following its approval in 2010. Use of BMAs in patients with mCSPC without a need for osteoporotic fracture prevention may constitute overuse.

2068 Background: The high frequency of financial relationships between the pharmaceutical industry and influential oncologists who author clinical practice guidelines may influence guideline recommendations. Therefore, we assessed the financial relationships held by NCCN Guidelines panelists before and after joining the panel, compared to those held by a matched set of oncologists. Methods: Membership of NCCN Guidelines panels for the 20 most common cancers was obtained from archival guidelines and linked manually to Open Payments records of industry payments. We identified physicians who newly joined an NCCN panel during the August 2013-December 2018 study period, and we included medical oncologists who had at least 1 year of Open Payments data before and after joining. These medical oncologists who joined an NCCN panel (panelists) were matched 1:2 to medical oncologists with the same gender, institutional affiliation, and medical school graduation year, who did not join an NCCN panel (non-panelists). The dollar value of industry payments was then calculated over the 1 year before (pre-join) and after (post-join) the date that each panelist joined. We used generalized linear models to assess differences in industry payments between the panelists and matched non-panelists in the pre-join period. We used difference-in-difference estimation (DiD) to assess whether joining an NCCN panel was associated with increased payments in the post-join period. Results: There were 54 panelists and 108 non-panelists (matched from 1447 eligible oncologists at NCCN institutions). Mean per-oncologist payments among panelists were greater than non-panelists in the pre-join period ($11,259 vs $3,427, p = 0.02). From the pre-join to post-join period there was a similar increase in mean per-oncologist payments among panelists and non-panelists ($2,236 vs. $1,569, DiD estimate +$667, p = 0.77). Conclusions: Medical oncologists who were selected to an NCCN Guidelines panel had greater financial ties to industry compared to peer oncologists who were not selected. This difference was present prior to joining; oncologists did not experience a greater increase in financial payments from industry in the 1-year period after joining an NCCN panel. These results suggest an opportunity to reduce the potential influence of industry in oncology clinical practice guidelines through the selection of guideline panelists with fewer ties to industry.

7071 Background: The cost of many cancer drugs is very high, but it is unclear if these costs are associated with commensurate improvement in outcomes. We aimed to assess the association between the cost of cancer treatments and their clinical benefit, using the NCCN Evidence Blocks value assessment framework. Methods: The NCCN Evidence Blocks include 4 measures of clinical benefit: Efficacy, Safety, Quality of Evidence, and Consistency of Evidence. The NCCN assigns scores on each measure ranging from 1 (least favorable) to 5 (most favorable). We obtained the NCCN Evidence Blocks scores as of December 31, 2018 for all recommended cancer treatments for the 30 most prevalent cancers in the US. For each treatment, we calculated total treatment costs (including drugs, administration fees, and supportive care medications) using Medicare reimbursement rates. We categorized treatments as either “time-limited” or “time-unlimited” according to whether their costs are best reflected as per full treatment course (often, adjuvant/neoadjuvant treatments) (time-limited) or per month of therapy (often, treatments for advanced disease) (time-unlimited). We used generalized estimating equations, with clustering within treatment indications, to estimate the association between Evidence Blocks scores and treatment costs, modeling the expected change in cost associated with a one-unit increase in the score on an Evidence Blocks measure. Results: There were 541 time-unlimited and 845 time-limited treatments. Among time-unlimited treatments, monthly treatment cost ranged from $4 to $64,630. Monthly treatment cost was positively associated with Efficacy ($3,036, 95%CI: $1,782, $4,289) and Quality of Evidence ($1,509, 95%CI: $171, $2,847) but negatively associated with Safety (-$1,470, 95%CI: -$2,790, -$151) and Consistency of Evidence (-$2,003, 95%CI -$3,420, -$586). Among time-limited treatments, cost per course of therapy ranged from $0 to $775,559, and no measure was significantly associated with cost. Evidence Blocks scores accounted for little of the variation in treatment cost (linear model R-squared = 0.10 for time-unlimited, and < 0.01 for time-unlimited). Conclusions: The association between NCCN Evidence Blocks measures and treatment cost was inconsistent, and accounted for little of the cost variation among treatments for the same indication. The clinical benefit of cancer treatments does not appear to be a primary determinant of treatment cost, suggesting that current pricing models may be inadequate to incentivize the development and utilization of high-value treatments.

Importance Significant controversy exists regarding whether physicians factor personal financial considerations into their clinical decision making. Within oncology, several reimbursement policies may incentivize physicians to increase health care use. Objective To evaluate whether the financial incentives presented by oncology reimbursement policies affect physician practice patterns. Evidence Review Studies evaluating an association between reimbursement incentives and changes in reimbursement policy on oncology care delivery were reviewed. Articles were identified systematically by searching PubMed/MEDLINE, Web of Science, Proquest Health Management, Econlit, and Business Source Premier. English-language articles focused on the US health care system that made empirical estimates of the association between a measurement of physician reimbursement/compensation and a measurement of delivery of cancer treatment services were included. The Risk of Bias in Non-Randomized Studies of Interventions tool was used to assess risk of bias. There were no date restrictions on the publications, and literature searches were finalized on February 14, 2018. Findings Eighteen studies were included. All were observational cohort studies, and most had a moderate risk of bias. Heterogeneity of reimbursement policies and outcomes precluded meta-analysis; therefore, a qualitative synthesis was performed. Most studies (15 of 18 [83%]) reported an association between reimbursement and care delivery consistent with physician responsiveness to financial incentives, although such an association was not identified in all studies. Findings consistently suggested that self-referral arrangements may increase use of radiotherapy and that profitability of systemic anticancer agents may affect physicians’ choice of drug. Findings were less conclusive as to whether profitability of systemic anticancer therapy affects the decision of whether to use any systemic therapy. Conclusions and Relevance To date, this study is the first systematic review of reimbursement policy and clinical care delivery in oncology. The findings suggest that some oncologists may, in certain circumstances, alter treatment recommendations based on personal revenue considerations. An implication of this finding is that value-based reimbursement policies may be a useful tool to better align physician incentives with patient need and increase the value of oncology care.

Background Financial relationships between physicians and the pharmaceutical industry are common, but factors that may determine whether such relationships result in physician practice changes are unknown. Materials and Methods We evaluated physician use of orally administered cancer drugs for four cancers: prostate (abiraterone, enzalutamide), renal cell (axitinib, everolimus, pazopanib, sorafenib, sunitinib), lung (afatinib, erlotinib), and chronic myeloid leukemia (CML; dasatinib, imatinib, nilotinib). Separate physician cohorts were defined for each cancer type by prescribing history. The primary exposure was the number of calendar years during 2013–2015 in which a physician received payments from the manufacturer of one of the studied drugs; the outcome was relative prescribing of that drug in 2015, compared with the other drugs for that cancer. We evaluated whether practice setting at a National Cancer Institute (NCI)-designated Comprehensive Cancer Center, receipt of payments for purposes other than education or research (compensation payments), maximum annual dollar value received, and institutional conflict-of-interest policies were associated with the strength of the payment-prescribing association. We used modified Poisson regression to control confounding by other physician characteristics. Results Physicians who received payments for a drug in all 3 years had increased prescribing of that drug (compared with 0 years), for renal cell (relative risk [RR] 1.81, 95% confidence interval [CI] 1.58–2.07), CML (RR 1.22, 95% CI 1.08–1.39), and lung (RR 1.69, 95% CI 1.58–1.82), but not prostate (RR 0.97, 95% CI 0.93–1.02). Physicians who received compensation payments or >$100 annually had increased prescribing compared with those who did not, but NCI setting and institutional conflict-of-interest policies were not consistently associated with the direction of prescribing change. Conclusion The association between industry payments and cancer drug prescribing was greatest among physicians who received payments consistently (within each calendar year). Receipt of payments for compensation purposes, such as for consulting or travel, and higher dollar value of payments were also associated with increased prescribing.

This study examines the association between oncologists’ receipt of payments from pharmaceutical manufacturers and drug selection in 2 situations where there are multiple treatment options.

Our understanding of new genes is often built upon the knowledge of well-characterized genes. One avenue toward revealing such connections involves creation of strains with mutations in two or more defined genes to permit genetic interaction analysis. Strain manipulations can yield unexpected mutations at loci outside the defined targeted genes. In this report, we describe a method for rapid validation of multigene mutants, thus allowing an appraisal of the contribution of the defined targeted genes to the strain’s phenotype., The biological function of a gene is often probed through its interactions with other genes. This general approach has been especially useful to build knowledge about poorly understood genes upon the bedrock of well-characterized genes. Genetic interaction analysis requires the construction of strains with mutations in two or more genes. Single-gene mutants of microbial pathogens are generally validated through introduction of a wild-type copy of the affected gene to create a complemented or reconstituted strain, followed by testing for restoration of a wild-type phenotype. This practice, formalized as one of Falkow’s “molecular Koch’s postulates” ensures that the phenotype of the mutant depends upon the known mutation. However, multigene mutants are seldom validated because of the labor required to assemble multiple genomic segments into a vector that can be introduced into the mutant strain. We present here an approach, concatemer assembly for rescue of mutant abilities (CARMA), that circumvents this impediment through an in vivo recombinational assembly strategy that does not require cloning at all. Our results show that CARMA allows genetic rescue of two double-gene mutant strains of the fungal pathogen Candida albicans. IMPORTANCE Our understanding of new genes is often built upon the knowledge of well-characterized genes. One avenue toward revealing such connections involves creation of strains with mutations in two or more defined genes to permit genetic interaction analysis. Strain manipulations can yield unexpected mutations at loci outside the defined targeted genes. In this report, we describe a method for rapid validation of multigene mutants, thus allowing an appraisal of the contribution of the defined targeted genes to the strain’s phenotype.

This study in a dimorphic fungal pathogen uncovered a role for a yeast-specific cytochrome P450 (CYP)-encoding gene in the ability of T. marneffei to grow as yeast cells within the host macrophages. This report will inspire further research into the role of CYPs and secondary metabolite synthesis during fungal pathogenic growth., Fungi are adept at occupying specific environmental niches and often exploit numerous secondary metabolites generated by the cytochrome P450 (CYP) monoxygenases. This report describes the characterization of a yeast-specific CYP encoded by simA ("survival in macrophages"). Deletion of simA does not affect yeast growth at 37°C in vitro but is essential for yeast cell production during macrophage infection. The ΔsimA strain exhibits reduced conidial germination and intracellular growth of yeast in macrophages, suggesting that the enzymatic product of SimA is required for normal fungal growth in vivo. Intracellular ΔsimA yeast cells exhibit cell wall defects, and metabolomic and chemical sensitivity data suggest that SimA may promote chitin synthesis or deposition in vitro. In vivo, ΔsimA yeast cells subsequently lyse and are degraded, suggesting that SimA may increase resistance to and/or suppress host cell biocidal effectors. The results suggest that simA synthesizes a secondary metabolite that allows T. marneffei to occupy the specific intracellular environmental niche within the macrophage. IMPORTANCE This study in a dimorphic fungal pathogen uncovered a role for a yeast-specific cytochrome P450 (CYP)-encoding gene in the ability of T. marneffei to grow as yeast cells within the host macrophages. This report will inspire further research into the role of CYPs and secondary metabolite synthesis during fungal pathogenic growth.

Although narrow eligibility criteria improve the internal validity of clinical trials, they may result in differences between study populations and real-world patients, threatening generalizability. Therefore, we evaluated whether patients treated for metastatic renal cell cancer (mRCC) in routine clinical practice are similar to those enrolled onto clinical trials.In this cohort study, we compared baseline characteristics of patients with mRCC in phase III clinical trials of new targeted therapies and those in a retrospective registry composed of academic (Duke) and community (ACORN Network) practices.A total of 438 registry patients received sunitinib, sorafenib, temsirolimus, or pazopanib (most commonly used agents) in first-line treatment. Registry patients receiving tyrosine kinase inhibitors (sunitinib, sorafenib, or pazopanib) were more likely to have poor-risk disease by Memorial Sloan Kettering Cancer Center criteria (poor, 7.4% v 2.9%; P.001; favorable, 30.1% v 43.8%; P.001) and to have impaired performance status (Eastern Cooperative Oncology Group1, 11.1% v 0.6%; P.001). However, registry patients receiving temsirolimus were less likely to have poor-risk disease (poor, 10.2% v 69.4%; P.001; favorable, 16.9% v 0%; P.001). Thus, 39.0% of registry patients would have been excluded from the phase III clinical trial testing the drug they received.Patients with mRCC treated with tyrosine kinase inhibitors in real-world clinical practice are sicker than those enrolled onto pivotal clinical trials, and more than one third are trial ineligible. Application of clinical trial findings to dissimilar populations may result in patient harm. Clinical research with more inclusive eligibility criteria is needed to appropriately guide real-world practice.

Recent changes in the Fungal Code of Nomenclature and developments in molecular phylogeny are about to lead to dramatic changes in the naming of medically important molds and yeasts. In this article, we present a widely supported and simple proposal to prevent unnecessary nomenclatural instability.

Candida glabrata is the second most common species of Candida recovered from patients with invasive candidiasis. The increasing number of infections due to C. glabrata, combined with its high rates of resistance to the commonly used, well-tolerated azole class of antifungal agents, has limited the use of this antifungal class. This has led to the preferential use of echinocandins as empirical treatment for serious Candida infections. The primary mechanism of resistance found in clinical isolates is the presence of an activating mutation in the gene encoding the transcription factor Pdr1 that results in upregulation of one or more of the efflux pumps Cdr1, Pdh1, and Snq2. By developing a better understanding of this mechanism of resistance to the azoles, it will be possible to develop strategies for reclaiming the utility of the azole antifungals against this important fungal pathogen., The high prevalence of fluconazole resistance among clinical isolates of Candida glabrata has greatly hampered the utility of fluconazole for the treatment of invasive candidiasis. Fluconazole resistance in this yeast is almost exclusively due to activating mutations in the transcription factor Pdr1, which result in upregulation of the ABC transporter genes CDR1, PDH1, and SNQ2 and therefore increased fluconazole efflux. However, the regulation of Pdr1 is poorly understood. In order to identify genes that interact with the Pdr1 transcriptional pathway and influence the susceptibility of C. glabrata to fluconazole, we screened a collection of deletion mutants for those exhibiting increased resistance to fluconazole. Deletion of the gene coding for a protein homologous to the Saccharomyces cerevisiae J protein Jjj1 resulted in decreased fluconazole susceptibility. We used the SAT1 flipper method to generate independent deletion mutants for JJJ1 in an SDD clinical isolate. Expression of both CDR1 and PDR1 was increased in the absence of JJJ1. In the absence of CDR1 or PDR1, deletion of JJJ1 has only a modest effect on fluconazole susceptibility. Transcriptional profiling using transcriptome sequencing (RNA-seq) revealed upregulation of genes of the Pdr1 regulon in the absence of JJJ1. Jjj1 appears to be a negative regulator of fluconazole resistance in C. glabrata and acts primarily through upregulation of the ABC transporter gene CDR1 via activation of the Pdr1 transcriptional pathway. IMPORTANCE Candida glabrata is the second most common species of Candida recovered from patients with invasive candidiasis. The increasing number of infections due to C. glabrata, combined with its high rates of resistance to the commonly used, well-tolerated azole class of antifungal agents, has limited the use of this antifungal class. This has led to the preferential use of echinocandins as empirical treatment for serious Candida infections. The primary mechanism of resistance found in clinical isolates is the presence of an activating mutation in the gene encoding the transcription factor Pdr1 that results in upregulation of one or more of the efflux pumps Cdr1, Pdh1, and Snq2. By developing a better understanding of this mechanism of resistance to the azoles, it will be possible to develop strategies for reclaiming the utility of the azole antifungals against this important fungal pathogen.

IntroductionThe rising cost of cancer drugs may make treatment unaffordable for some patients. Patients often rely on drug manufacturer-administered Pharmaceutical Assistance Programs (PAPs) to obtain drugs and reduced or no cost. The overall usage of PAPs within cancer care delivery is unknown.MethodsWe included all cancer patients across an academically affiliated, integrated health system in North Carolina during 2014 ( N = 8591). We identified the subset of patients receiving PAP assistance to afford one or more cancer drugs, in order to calculate the proportion of patients receiving PAP assistance, and the retail value of the assistance.ResultsAmong 8591 cancer patients, 215 unique patients submitted a total of 478 successful PAP requests for cancer drugs. 40% of PAP-utilizing patients were uninsured, 23% had Medicaid coverage, 20% had Medicare coverage, 2% were dual Medicare/Medicaid eligible, and 14% were commercially insured. Among all cancer patients who received medical treatment, 6.0% required PAP assistance, whereas 10.6% receiving an oral agent required PAP assistance. The proportion receiving PAP assistance varied substantially by drug, ranging from ConclusionsA substantial proportion of cancer patients receive private charitable assistance to obtain standard-of-care treatments. This includes patients with federal and private insurance, suggesting an inability of patients to meet cost-sharing requirements.

Candida albicans is a natural component of the human microbiota but also an opportunistic pathogen that causes life-threatening infections. The human gastrointestinal tract is the main reservoir of C. albicans, from where systemic infections originate as a consequence of the disruption of the intestinal mucosal barrier. Recent studies provided convincing evidence that overgrowth of C. albicans and other related species in the gut is predominantly associated with chronic intestinal inflammatory bowel diseases. Here, we showed, for the first time, the antagonistic interkingdom interactions between C. albicans and common intestinal commensal bacteria. From a therapeutic perspective, administering a defined bacterial community, such as the one described here with anti-Candida activity, could provide potential therapeutic protection against gastrointestinal inflammatory diseases., Candida albicans is well known as a major human fungal pathogen, but it is also a permanent resident of healthy gastrointestinal tracts. Recent studies have shown that the human gut microbial metabolome represents an interesting source of bioactive molecules with a significant degree of chemical diversity. Some of these bioactive molecules may have useful antivirulence activities. For instance, intestinal bacterial species belonging to the Lachnospiraceae family were found to secrete molecules that attenuate Salmonella pathogenicity and repress the expression of virulence genes. Here, we have investigated whether the microbial gut metabolome (GM) contains molecules that might promote the commensal lifestyle and/or inhibit the expression of virulence of C. albicans in the intestine. We found that metabolites from human feces inhibited the growth of C. albicans and other opportunistic yeasts. A genetic screen in C. albicans suggested that TOR is the molecular target of the antifungal molecule(s) of the GM. In addition, we found that the GM metabolites inhibit both C. albicans hyphal growth and the invasion of human enterocytes. The antigrowth and antivirulence activities were partially recapitulated by secretions from Roseburia spp. and Bacteroides ovatus strains, respectively. This study demonstrates that the antimicrobial activity of the GM can be extended to a eukaryotic pathogen, C. albicans, illuminating the antagonistic interkingdom interactions between a fungus and intestinal commensal bacteria. IMPORTANCE Candida albicans is a natural component of the human microbiota but also an opportunistic pathogen that causes life-threatening infections. The human gastrointestinal tract is the main reservoir of C. albicans, from where systemic infections originate as a consequence of the disruption of the intestinal mucosal barrier. Recent studies provided convincing evidence that overgrowth of C. albicans and other related species in the gut is predominantly associated with chronic intestinal inflammatory bowel diseases. Here, we showed, for the first time, the antagonistic interkingdom interactions between C. albicans and common intestinal commensal bacteria. From a therapeutic perspective, administering a defined bacterial community, such as the one described here with anti-Candida activity, could provide potential therapeutic protection against gastrointestinal inflammatory diseases.

This work describes the increased activity of a natural antifungal peptide in the presence of another antifungal peptide from a different family. This is termed antifungal synergy. Synergy is important for decreasing the amount of antifungal molecule needed to control the disease. Traditionally, naturally occurring antifungal molecules are assayed in isolation. Identification of synergistic interactions between antifungal peptides means that their activities in a complex biological system are likely to be different from what we observe when examining them individually. This study identified synergy between an antifungal peptide and a group of peptides that do not affect fungal growth in vitro. This provides the foundation for generation of transgenic plants with increased resistance to fungal disease and identification of antifungal accessory factors that enhance the activity of innate immune molecules but do not have an antifungal effect on their own., Defensins are a large family of small, cationic, cysteine-rich proteins that are part of the defense arsenal that plants use for protection against potentially damaging fungal infections. The plant defensin NaD1 from Nicotiana alata is a potent antifungal protein that inhibits growth and kills a variety of fungal pathogens that affect both plant and animal (human) hosts. Some serine protease inhibitors have also been reported to be antifungal molecules, while others have no inhibitory activity against fungi. Here we describe the synergistic activity of the plant defensin NaD1 with a selection of serine protease inhibitors against the plant pathogens Fusarium graminearum and Colletotrichum graminicola and the animal pathogen Candida albicans. The synergistic activity was not related to the protease inhibitory activity of these molecules but may arise from activation of fungal stress response pathways. The bovine pancreatic trypsin inhibitor (BPTI) displayed the most synergy with NaD1. BPTI also acted synergistically with several other antifungal molecules. The observation that NaD1 acts synergistically with protease inhibitors provides the foundation for the design of transgenic plants with improved resistance to fungal disease. It also supports the possibility of naturally occurring accessory factors that function to enhance the activity of innate immunity peptides in biological systems. IMPORTANCE This work describes the increased activity of a natural antifungal peptide in the presence of another antifungal peptide from a different family. This is termed antifungal synergy. Synergy is important for decreasing the amount of antifungal molecule needed to control the disease. Traditionally, naturally occurring antifungal molecules are assayed in isolation. Identification of synergistic interactions between antifungal peptides means that their activities in a complex biological system are likely to be different from what we observe when examining them individually. This study identified synergy between an antifungal peptide and a group of peptides that do not affect fungal growth in vitro. This provides the foundation for generation of transgenic plants with increased resistance to fungal disease and identification of antifungal accessory factors that enhance the activity of innate immune molecules but do not have an antifungal effect on their own.

The nose, throat, and skin over the subclavian and femoral veins are the body sites which harbor the bacteria which most commonly cause health care-associated infection. We assessed the effect of nursing home residence on the microbiota of these body sites in older adults. We found that the microbiota composition of the different body sites was similar between nursing home and community participants, but we identified differences in relative abundance levels. We found remarkable similarities in the bacterial communities of different body sites in older adults who lived in nursing homes compared to those in the community among people who had not been on antibiotics for the past 3 months. We also found that the femoral skin microbiota had evidence of stool contamination in the nursing home residents, providing a rationale for improved skin hygiene. Taken together, it appears that the health care environment does not alter the microbiota to the extent that antibiotics do., Our objective for this study was to characterize the microbial communities of the anterior nares (nose), posterior pharynx (throat), and skin of the femoral and subclavian areas in older adults from nursing homes and the community. Older adults (≥65 years) without antibiotic use for the past 3 months were recruited from nursing homes (NH; n = 16) and from the community (CB; n = 51). Specimens were taken from nose, throat, and skin sites for culture and bacterial profiling using 16S rRNA gene sequencing. We found that pathogenic Gram-negative rod (GNR) colonization on the femoral skin was higher in NH participants than CB participants; otherwise, there were no differences in GNR colonization at other body sites or in Staphylococcus aureus colonization at any body site. Bacterial community profiling demonstrated that the operational taxonomic unit compositions of the different body sites were similar between NH and CB participants, but the analysis identified differences in relative abundance levels. Streptococcus spp. were more abundant and Prevotella spp. were less abundant in the throats of NH participants than in throats of CB participants. Proteus, Escherichia coli, and Enterococcus were more abundant in NH participants on the femoral skin. We found a pattern of decreased abundance of specific Proteobacteria in NH participants at the anterior nares and at both skin sites. We concluded that bacterial communities were largely similar in diversity and composition within body sites between older adults without recent antibiotic use from NH compared to those from the community. Our findings support the rationale for improved hygiene in NH residents to reduce the transmission risk of antibiotic-resistant bacteria, such as Enterococcus spp. or Enterobacteriaceae. IMPORTANCE The nose, throat, and skin over the subclavian and femoral veins are the body sites which harbor the bacteria which most commonly cause health care-associated infection. We assessed the effect of nursing home residence on the microbiota of these body sites in older adults. We found that the microbiota composition of the different body sites was similar between nursing home and community participants, but we identified differences in relative abundance levels. We found remarkable similarities in the bacterial communities of different body sites in older adults who lived in nursing homes compared to those in the community among people who had not been on antibiotics for the past 3 months. We also found that the femoral skin microbiota had evidence of stool contamination in the nursing home residents, providing a rationale for improved skin hygiene. Taken together, it appears that the health care environment does not alter the microbiota to the extent that antibiotics do.

Streptococcus pneumoniae (pneumococcus) is a major human pathogen. It is a common colonizer of the human respiratory track, where it utilizes cell-cell communication systems to coordinate population-level behaviors. We reasoned that secreted peptides that are highly expressed during infection are pivotal for virulence. Thus, we used in silico pattern searches to define a pneumococcal secretome and analyzed the transcriptome of the clinically important PMEN1 lineage to identify which peptide-encoding genes are highly expressed in vivo. In this study, we characterized virulence peptide 1 (vp1), a highly expressed Gly-Gly peptide-encoding gene in chinchilla middle ear effusions. The vp1 gene is widely distributed across pneumococcus as well as encoded in related species. Studies in the chinchilla model of middle ear infection demonstrated that VP1 is a virulence determinant. The vp1 gene is positively regulated by a transcription factor from the Rgg family and its cognate SHP (short hydrophobic peptide). In vitro data indicated that VP1 promotes increased thickness and biomass for biofilms grown on chinchilla middle ear epithelial cells. Furthermore, the wild-type biofilm is restored with the exogenous addition of synthetic VP1. We conclude that VP1 is a novel streptococcal regulatory peptide that controls biofilm development and pneumococcal pathogenesis.

The impact of fungal pathogens on human health is devastating. One of the most pervasive fungal pathogens is Candida albicans, which kills ~40% of people suffering from bloodstream infections. Treatment of these infections is extremely difficult, as fungi are closely related to humans, and there are limited drugs that kill the fungus without host toxicity. The capacity of C. albicans to transition between yeast and filamentous forms is a key virulence trait. Thus, understanding the genetic pathways that regulate morphogenesis could provide novel therapeutic targets to treat C. albicans infections. Here, we establish the small molecule staurosporine as an inducer of filamentous growth. We unveil distinct regulatory circuitry required for staurosporine-induced filamentation that appears to be unique to this filament-inducing cue. Thus, this work highlights the fact that small molecules, such as staurosporine, can improve our understanding of the pathways required for key virulence programs, which may lead to the development of novel therapeutics., Protein kinases are key regulators of signal transduction pathways that participate in diverse cellular processes. In fungal pathogens, kinases regulate signaling pathways that govern drug resistance, stress adaptation, and pathogenesis. The impact of kinases on the fungal regulatory circuitry has recently garnered considerable attention in the opportunistic fungal pathogen Candida albicans, which is a leading cause of human morbidity and mortality. Complex regulatory circuitry governs the C. albicans morphogenetic transition between yeast and filamentous growth, which is a key virulence trait. Here, we report that staurosporine, a promiscuous kinase inhibitor that abrogates fungal drug resistance, also influences C. albicans morphogenesis by inducing filamentation in the absence of any other inducing cue. We further establish that staurosporine exerts its effect via the adenylyl cyclase Cyr1 and the cyclic AMP (cAMP)-dependent protein kinase A (PKA). Strikingly, filamentation induced by staurosporine does not require the known upstream regulators of Cyr1, Ras1 or Pkc1, or effectors downstream of PKA, including Efg1. We further demonstrate that Cyr1 is capable of activating PKA to enable filamentation in response to staurosporine through a mechanism that does not require degradation of the transcriptional repressor Nrg1. We establish that staurosporine-induced filamentation is accompanied by a defect in septin ring formation, implicating cell cycle kinases as potential staurosporine targets underpinning this cellular response. Thus, we establish staurosporine as a chemical probe to elucidate the architecture of cellular signaling governing fungal morphogenesis and highlight the existence of novel circuitry through which the Cyr1 and PKA govern a key virulence trait. IMPORTANCE The impact of fungal pathogens on human health is devastating. One of the most pervasive fungal pathogens is Candida albicans, which kills ~40% of people suffering from bloodstream infections. Treatment of these infections is extremely difficult, as fungi are closely related to humans, and there are limited drugs that kill the fungus without host toxicity. The capacity of C. albicans to transition between yeast and filamentous forms is a key virulence trait. Thus, understanding the genetic pathways that regulate morphogenesis could provide novel therapeutic targets to treat C. albicans infections. Here, we establish the small molecule staurosporine as an inducer of filamentous growth. We unveil distinct regulatory circuitry required for staurosporine-induced filamentation that appears to be unique to this filament-inducing cue. Thus, this work highlights the fact that small molecules, such as staurosporine, can improve our understanding of the pathways required for key virulence programs, which may lead to the development of novel therapeutics.

Cryptococcus neoformans and Candida albicans are two major human fungal pathogens and together account for over 1.4 million infections annually, with very high mortality rates. These fungi often infect immunocompromised individuals, such as HIV/AIDS patients. In an effort to identify novel drugs with antifungal activity, we have screened the Pathogen Box for compounds with anticryptococcal and anticandidal activities. This approach led to the discovery of a promising lead compound (MMV688271) with strong antifungal potency under nutrient-limited conditions., Human fungal pathogens cause over 2 million infections per year and are major drivers of morbidity and mortality. Cryptococcus neoformans and Candida albicans are two of the most common fungal pathogens of humans, together accounting for a staggering 1.4 million infections annually, with very high mortality rates. Patients with dysfunctional immune systems, such as individuals with HIV/AIDS, are particularly susceptible to fungal infections. Unfortunately, relatively few antifungal drugs are currently available and fungi frequently develop resistance, further complicating treatment approaches. In this study, we screened the Pathogen Box chemical library (Medicines for Malaria Venture, Switzerland) in an effort to identify novel antifungal compounds. This approach led to the discovery of a novel, highly potent antifungal agent with activity against both C. neoformans and C. albicans. Our initial study of the mechanism of action suggested that this novel compound prevents fungal proliferation by targeting the ability of C. neoformans to withstand stress at the plasma membrane and cell wall. Because this compound had previously been shown to have low toxicity for mammalian cells, we propose that it represents an attractive lead compound for further antifungal drug development. IMPORTANCE Cryptococcus neoformans and Candida albicans are two major human fungal pathogens and together account for over 1.4 million infections annually, with very high mortality rates. These fungi often infect immunocompromised individuals, such as HIV/AIDS patients. In an effort to identify novel drugs with antifungal activity, we have screened the Pathogen Box for compounds with anticryptococcal and anticandidal activities. This approach led to the discovery of a promising lead compound (MMV688271) with strong antifungal potency under nutrient-limited conditions.

Background. The pathogenesis of intra-abdominal candidiasis is poorly understood. Methods. Mice were intraperitoneally infected with Candida albicans (1 × 106 colony-forming units) and sterile stool. nanoString assays were used to quantitate messenger RNA for 145 C. albicans genes within the peritoneal cavity at 48 hours. Results. Within 6 hours after infection, mice developed peritonitis, characterized by high yeast burdens, neutrophil influx, and a pH of 7.9 within peritoneal fluid. Organ invasion by hyphae and early abscess formation were evident 6 and 24 hours after infection, respectively; abscesses resolved by day 14. nanoString assays revealed adhesion and responses to alkaline pH, osmolarity, and stress as biologic processes activated in the peritoneal cavity. Disruption of the highly-expressed gene RIM101, which encodes an alkaline-regulated transcription factor, did not impact cellular morphology but reduced both C. albicans burden during early peritonitis and C. albicans persistence within abscesses. RIM101 influenced expression of 49 genes during intra-abdominal candidiasis, including previously unidentified Rim101 targets. Overexpression of the RIM101-dependent gene SAP5, which encodes a secreted protease, restored the ability of a rim101 mutant to persist within abscesses. Conclusions. A mouse model of intra-abdominal candidiasis is valuable for studying pathogenesis and C. albicans gene expression. RIM101 contributes to persistence within intra-abdominal abscesses, at least in part through activation of SAP5.

The ascomycete Zymoseptoria tritici is the causal agent of Septoria leaf blotch on wheat. Disease control relies mainly on resistant wheat cultivars and on fungicide applications. The fungus displays a high potential to circumvent both methods. Resistance against all unisite fungicides has been observed over decades. A different type of resistance has emerged among wild populations with multidrug-resistant (MDR) strains. Active fungicide efflux through overexpression of the major facilitator gene MFS1 explains this emerging resistance mechanism. Applying a bulk-progeny sequencing approach, we identified in this study a 519-bp long terminal repeat (LTR) insert in the MFS1 promoter, a relic of a retrotransposon cosegregating with the MDR phenotype. Through gene replacement, we show the insert as a mutation responsible for MFS1 overexpression and the MDR phenotype. Besides this type I insert, we found two different types of promoter inserts in more recent MDR strains. Type I and type II inserts harbor potential transcription factor binding sites, but not the type III insert. Interestingly, all three inserts correspond to repeated elements present at different genomic locations in either IPO323 or other Z. tritici strains. These results underline the plasticity of repeated elements leading to fungicide resistance in Z. tritici and which contribute to its adaptive potential. IMPORTANCE Disease control through fungicides remains an important means to protect crops from fungal diseases and to secure the harvest. Plant-pathogenic fungi, especially Zymoseptoria tritici , have developed resistance against most currently used active ingredients, reducing or abolishing their efficacy. While target site modification is the most common resistance mechanism against single modes of action, active efflux of multiple drugs is an emerging phenomenon in fungal populations reducing additionally fungicides’ efficacy in multidrug-resistant strains. We have investigated the mutations responsible for increased drug efflux in Z. tritici field strains. Our study reveals that three different insertions of repeated elements in the same promoter lead to multidrug resistance in Z. tritici . The target gene encodes the membrane transporter MFS1 responsible for drug efflux, with the promoter inserts inducing its overexpression. These results underline the plasticity of repeated elements leading to fungicide resistance in Z. tritici .

Candida albicans is a human commensal and the causative agent of candidiasis, a potentially invasive and life-threatening infection. C. albicans experiences wide changes in pH during both benign commensalism (a common condition) and pathogenesis, and its morphology changes in response to this stimulus. Neutral pH is considered an activator of hyphal growth through Rim101, but the effect of low pH on other morphology-related pathways has not been extensively studied. We sought to determine the role of cyclic AMP signaling, a central regulator of morphology, in the sensing of pH. In addition, we asked broadly what cellular processes were altered by pH in both the presence and absence of this important signal integration system. We concluded that cAMP signaling is impacted by pH and that cAMP broadly impacts C. albicans physiology in both pH-dependent and -independent ways., Candida albicans behaviors are affected by pH, an important environmental variable. Filamentous growth is a pH-responsive behavior, where alkaline conditions favor hyphal growth and acid conditions favor growth as yeast. We employed filamentous growth as a tool to study the impact of pH on the hyphal growth regulator Cyr1, and we report that downregulation of cyclic AMP (cAMP) signaling by acidic pH contributes to the inhibition of hyphal growth in minimal medium with GlcNAc. Ras1 and Cyr1 are generally required for efficient hyphal growth, and the effects of low pH on Ras1 proteolysis and GTP binding are consistent with diminished cAMP output. Active alleles of ras1 do not suppress the hyphal growth defect at low pH, while dibutyryl cAMP partially rescues filamentous growth at low pH in a cyr1 mutant. These observations are consistent with Ras1-independent downregulation of Cyr1 by low pH. We also report that extracellular pH leads to rapid and prolonged decreases in intracellular pH, and these changes may contribute to reduced cAMP signaling by reducing intracellular bicarbonate pools. Transcriptomics analyses found that the loss of Cyr1 at either acidic or neutral pH leads to increases in transcripts involved in carbohydrate catabolism and protein translation and glycosylation and decreases in transcripts involved in oxidative metabolism, fluconazole transport, metal transport, and biofilm formation. Other pathways were modulated in pH-dependent ways. Our findings indicate that cAMP has a global role in pH-dependent responses, and this effect is mediated, at least in part, through Cyr1 in a Ras1-independent fashion. IMPORTANCE Candida albicans is a human commensal and the causative agent of candidiasis, a potentially invasive and life-threatening infection. C. albicans experiences wide changes in pH during both benign commensalism (a common condition) and pathogenesis, and its morphology changes in response to this stimulus. Neutral pH is considered an activator of hyphal growth through Rim101, but the effect of low pH on other morphology-related pathways has not been extensively studied. We sought to determine the role of cyclic AMP signaling, a central regulator of morphology, in the sensing of pH. In addition, we asked broadly what cellular processes were altered by pH in both the presence and absence of this important signal integration system. We concluded that cAMP signaling is impacted by pH and that cAMP broadly impacts C. albicans physiology in both pH-dependent and -independent ways.

Streptococcus pneumoniae (pneumococcus) is a leading cause of death and disease in children and elderly. Genetic variability among isolates from this species is high. These differences, often the product of gene loss or gene acquisition via horizontal gene transfer, can endow strains with new molecular pathways, diverse phenotypes, and ecological advantages. PMEN1 is a widespread and multidrug-resistant pneumococcal lineage. Using comparative genomics we have determined that a regulator-peptide signal transduction system, TprA2/PhrA2, was acquired by a PMEN1 ancestor and is encoded by the vast majority of strains in this lineage. We show that TprA2 is a negative regulator of a PMEN1-specific gene encoding a lanthionine-containing peptide (lcpA). The activity of TprA2 is modulated by its cognate peptide, PhrA2. Expression of phrA2 is density-dependent and its C-terminus relieves TprA2-mediated inhibition leading to expression of lcpA. In the pneumococcal mouse model with intranasal inoculation, TprA2 had no effect on nasopharyngeal colonization but was associated with decreased lung disease via its control of lcpA levels. Furthermore, the TprA2/PhrA2 system has integrated into the pneumococcal regulatory circuitry, as PhrA2 activates TprA/PhrA, a second regulator-peptide signal transduction system widespread among pneumococci. Extracellular PhrA2 can release TprA-mediated inhibition, activating expression of TprA-repressed genes in both PMEN1 cells as well as another pneumococcal lineage. Acquisition of TprA2/PhrA2 has provided PMEN1 isolates with a mechanism to promote commensalism over dissemination and control inter-strain gene regulation., Author summary Streptococcus pneumoniae (pneumococcus), an important human pathogen, exhibits a dual lifestyle featuring asymptomatic colonization of the host on the one hand as well as infliction of severe local and systemic disease on the other. In pneumococcal strains, differences in gene possession often lead to varied phenotypic outcomes. Epidemiologically, pandemic strains of the PMEN1 lineage show high prevalence in disease as well as carriage, posing an interesting question on the composition and function of the genomic toolkit that leads to their widespread success. Here, we characterize TprA2/PhrA2 sensory system, a genomic region acquired exclusively by the PMEN1 strains. The system consists of a regulator-peptide pair that was horizontally acquired into PMEN1 along with its regulatory circuitry. The regulatory peptide PhrA2 is receptive to cell density of PMEN1 cells and is an example of elegant communication signaling between bacterial cells. The regulatory influence of PhrA2 extends beyond PMEN1 cells such that it controls genes of a widespread signaling system and virulence regulon in non-PMEN1 strains. This work contributes to the knowledge of peptide-communication signals in pneumococcus and further adds a novel mechanism by which an ecologically successful linage may modify the transcriptomic and functional landscape of a multi-strain pneumococcal community.

We find here that Ccr4, Pop2, and Dhh1 modulate the levels of mRNAs for specific Rho1 regulators, Rom2 and Lrg1. In budding yeast, Rho1 activity is tightly regulated both temporally and spatially. It is anticipated that Ccr4, Pop2, and Dhh1 may contribute to the precise spatiotemporal control of Rho1 activity by regulating expression of its regulators temporally and spatially. Our finding on the roles of the components of the Ccr4-Not complex in yeast would give important information for understanding the roles of the evolutionary conserved Ccr4-Not complex., Ccr4, a component of the Ccr4-Not cytoplasmic deadenylase complex, is known to be required for the cell wall integrity (CWI) pathway in the budding yeast Saccharomyces cerevisiae. However, it is not fully understood how Ccr4 and other components of the Ccr4-Not complex regulate the CWI pathway. Previously, we showed that Ccr4 functions in the CWI pathway together with Khd1 RNA binding protein. Ccr4 and Khd1 modulate a signal from Rho1 small GTPase in the CWI pathway by regulating the expression of ROM2 mRNA and LRG1 mRNA, encoding a guanine nucleotide exchange factor (GEF) and a GTPase-activating protein (GAP) for Rho1, respectively. Here we examined the possible involvement of the POP2 gene encoding a subunit of the Ccr4-Not complex and the DHH1 gene encoding a DEAD box RNA helicase that associates with the Ccr4-Not complex in the regulation of ROM2 and LRG1 expression. Neither ROM2 mRNA level nor Rom2 function was impaired by pop2Δ or dhh1Δ mutation. The LRG1 mRNA level was increased in pop2Δ and dhh1Δ mutants, as well as the ccr4Δ mutant, and the growth defects caused by pop2Δ and dhh1Δ mutations were suppressed by lrg1Δ mutation. Our results suggest that LRG1 expression is regulated by Ccr4 together with Pop2 and Dhh1 and that ROM2 expression is regulated by Khd1 and Ccr4, but not by Pop2 and Dhh1. Thus, Rho1 activity in the CWI pathway is precisely controlled by modulation of the mRNA levels for Rho1-GEF Rom2 and Rho1-GAP Lrg1. IMPORTANCE We find here that Ccr4, Pop2, and Dhh1 modulate the levels of mRNAs for specific Rho1 regulators, Rom2 and Lrg1. In budding yeast, Rho1 activity is tightly regulated both temporally and spatially. It is anticipated that Ccr4, Pop2, and Dhh1 may contribute to the precise spatiotemporal control of Rho1 activity by regulating expression of its regulators temporally and spatially. Our finding on the roles of the components of the Ccr4-Not complex in yeast would give important information for understanding the roles of the evolutionary conserved Ccr4-Not complex.

The yeast Candida albicans causes human infections that have mortality rates approaching 50%. The key to developing improved therapeutics is to understand the host-pathogen interface. A critical interaction is that with macrophages: intracellular Candida triggers the NLRP3/caspase-1 inflammasome for escape through lytic host cell death, but this also activates antifungal responses. To better understand how the inflammasome response to Candida is fine-tuned, we established live-cell imaging of inflammasome activation at single-cell resolution, coupled with analysis of the fungal ERMES complex, a mitochondrial regulator that lacks human homologs. We show that ERMES mediates Candida escape via inflammasome-dependent processes, and our data suggest that inflammasome activation is controlled by the level of hyphal growth and exposure of cell wall components as a proxy for severity of danger. Our study provides the most detailed dynamic analysis of inflammasome responses to a fungal pathogen so far and establishes promising pathogen- and host-derived therapeutic strategies., The pathogenic yeast Candida albicans escapes macrophages by triggering NLRP3 inflammasome-dependent host cell death (pyroptosis). Pyroptosis is inflammatory and must be tightly regulated by host and microbe, but the mechanism is incompletely defined. We characterized the C. albicans endoplasmic reticulum (ER)-mitochondrion tether ERMES and show that the ERMES mmm1 mutant is severely crippled in killing macrophages despite hyphal formation and normal phagocytosis and survival. To understand dynamic inflammasome responses to Candida with high spatiotemporal resolution, we established live-cell imaging for parallel detection of inflammasome activation and pyroptosis at the single-cell level. This showed that the inflammasome response to mmm1 mutant hyphae is delayed by 10 h, after which an exacerbated activation occurs. The NLRP3 inhibitor MCC950 inhibited inflammasome activation and pyroptosis by C. albicans, including exacerbated inflammasome activation by the mmm1 mutant. At the cell biology level, inactivation of ERMES led to a rapid collapse of mitochondrial tubular morphology, slow growth and hyphal elongation at host temperature, and reduced exposed 1,3-β-glucan in hyphal populations. Our data suggest that inflammasome activation by C. albicans requires a signal threshold dependent on hyphal elongation and cell wall remodeling, which could fine-tune the response relative to the level of danger posed by C. albicans. The phenotypes of the ERMES mutant and the lack of conservation in animals suggest that ERMES is a promising antifungal drug target. Our data further indicate that NLRP3 inhibition by MCC950 could modulate C. albicans-induced inflammation. IMPORTANCE The yeast Candida albicans causes human infections that have mortality rates approaching 50%. The key to developing improved therapeutics is to understand the host-pathogen interface. A critical interaction is that with macrophages: intracellular Candida triggers the NLRP3/caspase-1 inflammasome for escape through lytic host cell death, but this also activates antifungal responses. To better understand how the inflammasome response to Candida is fine-tuned, we established live-cell imaging of inflammasome activation at single-cell resolution, coupled with analysis of the fungal ERMES complex, a mitochondrial regulator that lacks human homologs. We show that ERMES mediates Candida escape via inflammasome-dependent processes, and our data suggest that inflammasome activation is controlled by the level of hyphal growth and exposure of cell wall components as a proxy for severity of danger. Our study provides the most detailed dynamic analysis of inflammasome responses to a fungal pathogen so far and establishes promising pathogen- and host-derived therapeutic strategies.

Background & Aims Immune cells of the liver must be able to recognize and react to pathogens yet remain tolerant to food molecules and other nonpathogens. Dendritic cells (DCs) are believed to contribute to hepatic tolerance. Lipids have been implicated in dysfunction of DCs in cancer. Therefore, we investigated whether high lipid content in liver DCs affects induction of tolerance. Methods Mouse and human hepatic nonparenchymal cells were isolated by mechanical and enzymatic digestion. DCs were purified by fluorescence-activated cell sorting or with immunomagnetic beads. DC lipid content was assessed by flow cytometry, immune fluorescence, and electron microscopy and by measuring intracellular component lipids. DC activation was determined from surface phenotype and cytokine profile. DC function was assessed in T-cell, natural killer (NK) cell, and NKT cell coculture assays as well as in vivo. Results We observed 2 distinct populations of hepatic DCs in mice and humans based on their lipid content and expression of markers associated with adipogenesis and lipid metabolism. This lipid-based dichotomy in DCs was unique to the liver and specific to DCs compared with other hepatic immune cells. However, rather than mediate tolerance, the liver DC population with high concentrations of lipid was immunogenic in multiple models; they activated T cells, NK cells, and NKT cells. Conversely, liver DCs with low levels of lipid induced regulatory T cells, anergy to cancer, and oral tolerance. The immunogenicity of lipid-rich liver DCs required their secretion of tumor necrosis factor α and was directly related to their high lipid content; blocking DC synthesis of fatty acids or inhibiting adipogenesis (by reducing endoplasmic reticular stress) reduced DC immunogenicity. Conclusions Human and mouse hepatic DCs are composed of distinct populations that contain different concentrations of lipid, which regulates immunogenic versus tolerogenic responses in the liver.

The cell wall is essential for viability of fungi and is an effective drug target in pathogens such as Candida albicans. The contribution of post-transcriptional gene regulators to cell wall integrity in C. albicans is unknown. We show that the C. albicans Ccr4-Pop2 mRNA deadenylase, a regulator of mRNA stability and translation, is required for cell wall integrity. The ccr4/pop2 mutants display reduced wall β-glucans and sensitivity to the echinocandin caspofungin. Moreover, the deadenylase mutants are compromised for filamentation and virulence. We demonstrate that defective cell walls in the ccr4/pop2 mutants are linked to dysfunctional mitochondria and phospholipid imbalance. To further understand mitochondrial function in cell wall integrity, we screened a Saccharomyces cerevisiae collection of mitochondrial mutants. We identify several mitochondrial proteins required for caspofungin tolerance and find a connection between mitochondrial phospholipid homeostasis and caspofungin sensitivity. We focus on the mitochondrial outer membrane SAM complex subunit Sam37, demonstrating that it is required for both trafficking of phospholipids between the ER and mitochondria and cell wall integrity. Moreover, in C. albicans also Sam37 is essential for caspofungin tolerance. Our study provides the basis for an integrative view of mitochondrial function in fungal cell wall biogenesis and resistance to echinocandin antifungal drugs.

6607Background: Physicians who accept payments from a pharmaceutical company are more likely to prescribe that company’s cancer drug[s]. Whether long-term physician-industry relationships are more ...

Yeast cells contain two Bro1 domain proteins: Bro1, which is required for endosomal trafficking, and Rim20, which is required for the response to the external pH via the Rim101 pathway. Rim20 associates with endosomal structures under alkaline growth conditions, when it promotes activation of Rim101 through proteolytic cleavage. We report here that the pH-dependent localization of Rim20 is contingent on the amount of Bro1 in the cell. Cells that lack Bro1 have increased endosomal Rim20-green fluorescent protein (GFP) under acidic conditions; cells that overexpress Bro1 have reduced endosomal Rim20-GFP under acidic or alkaline conditions. The novel endosomal association of Rim20-GFP in the absence of Bro1 requires ESCRT components including Vps27 but not specific Rim101 pathway components such as Dfg16. Vps27 influences the localization of Bro1 but is not required for RIM101 pathway activation in wild-type cells, thus suggesting that Rim20 enters the Bro1 localization pathway when a vacancy exists. Despite altered localization of Rim20, the lack of Bro1 does not bypass the need for signaling protein Dfg16 to activate Rim101, as evidenced by the expression levels of the Rim101 target genes RIM8 and SMP1 . Therefore, endosomal association of Rim20 is not sufficient to promote Rim101 activation.

It has come to our attention that approximately 35% of >100 published micro-array data sets, where transcript levels were compared between two different strains, exhibit some form of chromosome-specific bias. While some of these arose from the use of strains whose aneuploidies were not known at the time, in a worrisome number of cases the recombinant strains have acquired additional aneuploidies that were not initially present in the parental strain. The aneuploidies often affected a different chromosome than the one harboring the insertion site. The affected strains originated from either CAI-4, RM1000, BWP17 or SN95 and were produced through a variety of strategies. These observations suggest that aneuploidies frequently occur during the production of recombinant strains and have an effect on global transcript profiles outside of the afflicted chromosome(s), thus raising the possibility of unintended phenotypic consequences. Thus, we propose that all Candida albicans mutants and strains should be tested for aneuploidy before being used in further studies. To this end, we describe a new rapid testing method, based on a multiplex quantitative PCR assay, that produces eight bands of distinct sizes from either the left or right arms of each C. albicans chromosome.

The cell wall of Candida albicans lies at the crossroads of pathogenicity and therapeutics. It contributes to pathogenicity through adherence and invasion; it is the target of both chemical and immunological antifungal strategies. We have initiated a dissection of cell wall function through targeted insertional mutagenesis of cell wall-related genes. Among 25 such genes, we were unable to generate homozygous mutations in 4, and they may be essential for viability. We created homozygous mutations in the remaining 21 genes. Insertion mutations in SUN41 , Orf19.5412, Orf19.1277, MSB2 , Orf19.3869, and WSC1 caused hypersensitivity to the cell wall inhibitor caspofungin, while two different ecm33 insertions caused mild caspofungin resistance. Insertion mutations in SUN41 and Orf19.5412 caused biofilm defects. Through analysis of homozygous sun41 Δ/ sun41 Δ deletion mutants and sun41 Δ/ sun41 Δ+p SUN41 -complemented strains, we verified that Sun41 is required for biofilm formation and normal caspofungin tolerance. The sun41 Δ/ sun41 Δ mutant had altered expression of four cell wall damage response genes, thus suggesting that it suffers a cell wall structural defect. Sun41 is required for inducing disease, because the mutant was severely attenuated in mouse models of disseminated and oropharyngeal candidiasis. Although the mutant produced aberrant hyphae, it had no defect in damaging endothelial or epithelial cells, unlike many other hypha-defective mutants. We suggest that the sun41 Δ/ sun41 Δ cell wall defect is the primary cause of its attenuated virulence. As a small fungal surface protein with predicted glucosidase activity, Sun41 represents a promising therapeutic target.